The Tet-on system for controllable gene expression in the rock-inhabiting black fungus Knufia petricola

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K. petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K. petricola cell or for using K. petricola as host for the expression of heterologous genes. Supplementary Information The online version contains supplementary material available at 10.1007/s00792-024-01354-2.

(a) Five intergenic regions on four different contigs were chosen.Igr1 and igr2 have been validated previously (Erdmann et al. 2022).Three additional regions on other contigs of the K. petricola A95 genome assembly were chosen.Shown are the insertion sites (at Cas9-mediated DSBs via igr-specific PS1), homologous sequences contained in donor DNA (orange -short homologous (SH) sequences flanking PCR-generated donor DNA, light orange -long homologous (LH) sequences flanking constructs assembled in pIGR1/2R(-TGG) cloning vectors), and primers used for detection of targeted insertion by homologous recombination (HR).(b) Strategies for targeted integration of expression constructs.Cloning vectors containing LH sequences to igr1 or igr2 [pIGRXR(-XXX) series] can be used for the assembly of expression constructs for targeted integration.Expression constructs with SH sequences for the insertion in all igrs can be generated by PCR using appropriate primers and templates.Exemplarily shown is the insertion of expression constructs with SH sequences derived from pNXR-XXX cloning vectors, which carry the resistance (R) and expression (E) cassette in an inverse orientation.The rtTA cassette for the expression of the DOX-regulated transcription factor is present in TET constructs only.(c) Identification of transformants carrying a TET::gfp construct at igr2.The [(TniaD::hph::PtrpC)-(PoliC::rtTA::TcrgA)-(Ptet::gfp::Tgluc)] expression construct flanked by 75-bp-long homologous sequences to igr2 was amplified from pNAH-OTGG using the primers TniiA-igr2-SH5F and Tgluc-igr2-SH3R.Protoplasts of WT:A95 were transformed with the construct as donor DNA and the Cas9-and sgRNA-delivering pAMA/ribo-igr2 PS1 (Table S7).By diagnostic PCR using primers binding to the ends of the R or E cassette and in igr2, four strains with correctly inserted expression constructs were identified.S6).Genes of interest (goi) are inserted upstream of gfp for C-terminal GFP, downstream of gfp for N-terminal GFP or by replacing gfp using the restriction sites NcoI or NotI for linearization of the entry plasmid.Donor DNA for transformation of K. petricola i.e., linear expression constructs flanked by homologous sequences, can be generated by digestion of the plasmids with restriction enzymes cutting in the multiple cloning sites (MCS), by amplification with short primers igrX-RF-F1 and igrX-RF-R1, or by using primers binding to the terminators (TniaD-xxx-SH5F, Tgluc-xxx-SH3R) and containing the 75-bp-long 5' overhangs for attaching SH sequences (shown as orange boxes).Further abbreviations: 5FA, 3RA -synthetic adapter sequences in the pIGRXR series; R gene -hph, nat1, nptII, bar and sur for mediating resistance against hygromycin B (HYG), nourseothricin (NTC), geneticin (G418), glufosinate (GFS) and chlorimuron ethyl (CME), respectively; POI -protein of interest.(b) Vectors for cloning constructs for bimolecular fluorescence complementation (BiFC) studies.The four vectors containing either a hygR cassette and the LH sequences of igr1 or a natR cassette and the LH sequences of igr2 were cloned using members of the pIGRXR-TGG series as entry plasmids (Table S6).GfpC-linker fragments were amplified from pNAH-OGCGn/c (Schumacher 2012) and assembled in NcoI+NotI-digested pIGR1H-TGG yielding pIGR1H-TGCGn and pIGR1H-TGCGc.For pIGR2N-TGNTn and pIGR2N-TGNTc, the gfpN-linker fragments were amplified with TtrpC from pNDN-AGNTn/c (Schumacher 2012) and assembled in NcoI+PacI-digested pIGR2N-TGG.Note that the linker (L) sequences differ, and that the used gfp sequence contains an intron in its 5' region (light green box).The sites for insertion of a goi -with stop codon for N-terminal GFP-Linker (n) and without stop codon for C-terminal Linker-GFP (c) -are indicated by gray arrows.(a) Verification of the integration of the three constructs in igr1, igr2, and igr3, respectively.Four HYG-, NTC-and G418-resistant transformants of OE::WCC black and TET::WCC black strains were screened by diagnostic PCR using primers binding to the ends of the constructs and in igrX.Amplicons for HR and the absence of the wild type intergenic regions verified the correct insertions.(b) Identification of optimal DOX concentration and induction time for protein-protein interaction studies.Two-day-old liquid cultures of OE::WCC black (T1) and TET::WCC black (T2) were split in five one ml cultures for incubation without and with 25, 50, 75 and 100 µg/ml DOX. 100 µl of the cultures were sampled at the indicated time points and directly used for fluorescence microscopy with exposure times of 80 ms (mCH) and 800 ms (GFP).Scale bar -10 µm.Restriction sites for the rarely cutting enzymes AscI and PacI (sequences in blue) and start (ATG) and stop codons (TAA) were inserted up-and downstream of the GSG-P2A sequences.One or more genes can be inserted.This entry plasmid also allows for testing the effect of the attached P2A on the activity of a single protein by inserting the gene upstream of the P2A motif.p426 GAL1 is an E. coli/S.cerevisiae shuttle vector containing the full-length S. cerevisiae GAL1 promoter and CYC1 terminator which flank a cloning site for insertion of a goi, and URA3 and the 2-micron for selection and propagation in S. cerevisiae (Mumberg et al. 1994).(c) The four plasmids for the expression of two or three carotenogenic genes were cloned in several steps.Cloning involved the generation of several intermediates and was accomplished by the digestion of an entry plasmid (in gray boxes) and its assembly with amplicons (primers and templates are shown), single-stranded (ss) or double-stranded (ds) synthetic DNA (see Table S6).K. petricola genes were amplified in the first step from cDNA to obtain intron-free genes for the expression in S. cerevisiae.The DNA assembly was performed in S. cerevisiae (in vivo) or in vitro.pIGR2H-TET::(G)PP containing the intron-free variants of the carotenogenic K. petricola genes carry the 2-micron and S. cerevisiae URA3 in their backbones.Consequently, they may mediate episomal gene expression in S. cerevisiae, as far as the synthetic transactivator-encoding gene is expressed.For control, yeast expression vectors were cloned (pURA-GAL::(G)PP) that contained the fused carotenogenic genes downstream of the S. cerevisiae GAL1 promoter for galactose-inducible gene expression (Fig. S6c).The plasmid-carrying S. cerevisiae strains were cultivated for three days on selective medium (SD-URA) containing 2 % glucose (GLU) or 4 % galactose (GAL) and/or 10 µg/ml DOX.
Fig S2 Cloning of TET-containing expression constructs for integration in K. petricola igr1 or igr2.(a)The pIGRXR-TGG series for TET-regulated expression of a gene or a gene-gfp fusion protein.Vectors were generated as follows: the TGG fragment shown was amplified by PCR from pNAH-OTGG(Janevska et al. 2017) with primers containing 5' overhangs to PtrpC and the 3R-adapter, and assembled with PacI-digested pIGRXR plasmids(Erdmann et al. 2022).In sum, twelve cloning vectors were cloned containing different resistance (R) genes flanked by A. nidulans PtrpC and B. cinerea TniaD, and homologous sequences to igr1 or igr2 (TableS6).Genes of interest (goi) are inserted upstream of gfp for C-terminal GFP, downstream of gfp for N-terminal GFP or by replacing gfp using the restriction sites NcoI or NotI for linearization of the entry plasmid.Donor DNA for transformation of K. petricola i.e., linear expression constructs flanked by homologous sequences, can be generated by digestion of the plasmids with restriction enzymes cutting in the multiple cloning sites (MCS), by amplification with short primers igrX-RF-F1 and igrX-RF-R1, or by using primers binding to the terminators (TniaD-xxx-SH5F, Tgluc-xxx-SH3R) and containing the 75-bp-long 5' overhangs for attaching SH sequences (shown as orange boxes).Further abbreviations: 5FA, 3RA -synthetic adapter sequences in the pIGRXR series; R gene -hph, nat1, nptII, bar and sur for mediating resistance against hygromycin B (HYG), nourseothricin (NTC), geneticin (G418), glufosinate (GFS) and chlorimuron ethyl (CME), respectively; POI -protein of interest.(b) Vectors for cloning constructs for bimolecular fluorescence complementation (BiFC) studies.The four vectors containing either a hygR cassette and the LH sequences of igr1 or a natR cassette and the LH sequences of igr2 were cloned using members of the pIGRXR-TGG series as entry plasmids (TableS6).GfpC-linker fragments were amplified from pNAH-OGCGn/c(Schumacher 2012) and assembled in NcoI+NotI-digested pIGR1H-TGG yielding pIGR1H-TGCGn and pIGR1H-TGCGc.For pIGR2N-TGNTn and pIGR2N-TGNTc, the gfpN-linker fragments were amplified with TtrpC from pNDN-AGNTn/c (Schumacher 2012) and assembled in NcoI+PacI-digested pIGR2N-TGG.Note that the linker (L) sequences differ, and that the used gfp sequence contains an intron in its 5' region (light green box).The sites for insertion of a goi -with stop codon for N-terminal GFP-Linker (n) and without stop codon for C-terminal Linker-GFP (c) -are indicated by gray arrows.

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Fig S3 Igr3, igr4, and igr5 are additional neutral insertion sites in the K. petricola genome.Strains with insertions at igr3, igr4 or igr5 were generated by transformation of WT:A95 protoplasts with pAMA/tRNA-igrX PS1 and [(TniaD::hph::PtrpC)-(PoliC::gfp::Tgluc)] expression constructs flanked by 75-bp-long homologous sequences to igrX.Strains containing the same expression constructs in igr1 and igr2 were available from a previous study.(a) Strains with targeted integration of the GFP expression constructs were identified by diagnostic PCRs.Combinations of primers binding in the igrX or the construct (Fig S1) were used for detecting HR events and the absence of the wild type locus.(b) GFP is equally well expressed from all five intergenic regions.Cells of two-day-old liquid cultures were dropped onto objective slides and submitted to fluorescence microscopy.Images were captured with exposure times of 40 ms.Mean values and standard deviations of GFP fluorescence intensities derived from 100 cells of two transformants per strain and 50 cells of WT. * p <0.001 compared to WT. (c) Insertions in the intergenic regions do not affect growth under different stress conditions.Cells (10 4 , 10 3 , 10 2 , 10 1 ) were dropped onto solid SDNG pH 5 (control) and modified media as shown.Pictures were taken 10 dpi.
Fig S5 Generation of K. petricola Δpks1/Δphs1-phd1 mutants.(a) Strategies for simultaneous replacement of pks1, phs1 and phd1.Protoplasts of WT:A95 were co-transformed with the Cas9-and sgRNA delivering plasmid pAMA/tRNA-pks1 PS2 -phs1 PS1 and resistance cassette-containing donor DNA with 75-bp-long homologous sequences to the non-coding regions up-and downstream of pks1 or phs1-phd1 (TableS7).Pks1 was replaced by a natR cassette, the physically linked phs1 and phd1 by a glufosinate resistance (baR) cassette.The location of Cas9 sites (protospacers, PS) for introduction of DSBs in the coding regions of pks1 and phs1 are shown.(b) Diagnostic PCRs confirmed the replacement of pks1 and physically linked phs1 and phd1 in three transformants.Six resistant transformants were screened by PCR for HR events and the absence of the genes of interest (ORF).Primers binding up-or downstream of genes of interest or to the resistance cassettes were used as shown in a. Transformants T1, T4, and T6 exhibit the expected amplicon pattern.(c) The three mutants exhibit an albino phenotype.Wild type A95, the newly generated mutants and available mutants defective in melanogenesis (Δpks1) or melanogenesis and carotenogenesis (Δpks1/Δphs1) for comparison were cultivated for ten days on solidified MEA and SDNG.
Fig S7 Expression of the carotenogenic K. petricola genes from pIGRXR-TET in S. cerevisiae.pIGR2H-TET::(G)PPcontaining the intron-free variants of the carotenogenic K. petricola genes carry the 2-micron and S. cerevisiae URA3 in their backbones.Consequently, they may mediate episomal gene expression in S. cerevisiae, as far as the synthetic transactivator-encoding gene is expressed.For control, yeast expression vectors were cloned (pURA-GAL::(G)PP) that contained the fused carotenogenic genes downstream of the S. cerevisiae GAL1 promoter for galactose-inducible gene expression (Fig.S6c).The plasmid-carrying S. cerevisiae strains were cultivated for three days on selective medium (SD-URA) containing 2 % glucose (GLU) or 4 % galactose (GAL) and/or 10 µg/ml DOX.